Chain flexibility of medicinal lipids determines their selective partitioning into lipid droplets

In guiding lipid droplets (LDs) to serve as storage vessels that insulate high-value lipophilic compounds in cells, we demonstrate that chain flexibility of lipids determines their selective migration in intracellular LDs. Focusing on commercially important medicinal lipids with biogenetic similarity but structural dissimilarity, we computationally and experimentally validate that LD remodeling should be differentiated between overproduction of structurally flexible squalene and that of rigid zeaxanthin and β-carotene. In molecular dynamics simulations, worm-like flexible squalene is readily deformed to move through intertwined chains of triacylglycerols in the LD core, whereas rod-like rigid zeaxanthin is trapped on the LD surface due to a high free energy barrier in diffusion. By designing yeast cells with either much larger LDs or with a greater number of LDs, we observe that intracellular storage of squalene significantly increases with LD volume expansion, but that of zeaxanthin and β-carotene is enhanced through LD surface broadening; as visually evidenced, the outcomes represent internal penetration of squalene and surface localization of zeaxanthin and β-carotene. Our study shows the computational and experimental validation of selective lipid migration into a phase-separated organelle and reveals LD dynamics and functionalization.

The TAG synthesis-related proteins, such as Lro1 and Dga1, in the ER membrane devote to the lens formation (see yellow box). Additionally, fatty acids (FAs) which are important sources of neutral lipids can be produced by mitochondrial cardiolipin-specific phospholipase, Cld1.
When the concentration of neutral lipids exceeds a certain threshold, LDs bud toward cytosol.
The Sei1, Loa1 and Erd1 proteins promote detachment of LDs from ER. The mature LD can be degraded by lipolysis and β-oxidation. The Tgl3, Tgl4, Tgl5, and Pex10 are involved in TAG degradation. The proteins related to increase size and number indicate color-coded in blue and red, respectively. and non-producing control cells (B). β-carotene (red) was favorably accommodated on all kinds of membranes including the LD surface (green). The cells were grown in YSC medium with 2% (w/v) glucose at 30 °C. We note that β-carotene was inherently fluorescent (excitation at 450 nm and emission at 600 nm), and LD was stained with the BODIPY fluorescent dye (excitation at 488 nm and emission at 500 nm). The confocal fluorescence microscopy experiments were performed at least triplicate at two independent times. Scale bar, 5 µm.  The ratio of the amount of production by the engineered cells for each approach to that by the non-engineered cells. 8

Supplementary Note 1. Determination of key genes in creating two different designer yeast cells featuring large LD size and number
Lipid droplets (LDs) are highly dynamic organelles, and alternating between their biogenesis and degradation can cause the dramatic change in LD number and size. The LD biogenesis and degradation are controlled by the enzymes that promote synthesis and hydrolysis of neutral lipids and proteins as involved in budding of LDs from endoplasmic reticulum (ER) (see Figure 3A and Supplementary Figure 2 We carefully identified ten different genes of which effects on the LD number or size have been validated by previous studies (Table 1), and each gene was deleted or overexpressed in a wild-type (WT) yeast strain, enabling comprehensive evaluation of its specific effect on the LD number and size (see Figure 3B, Supplementary Figure 3, and Supplementary Table   1). Similar with previous studies of LD number and size 2,3 , the cells were grown in YSC medium with 2% (w/v) glucose at 30 °C for 24 h, stained with BODIPY dye (excitation at 488 nm and emission at 500 nm), and examined by confocal fluorescence microscopy.
Approximately, ~300 LDs for every strain were investigated for the statistical analysis of LD number and size.
In terms of increasing the size of LDs, we focused on enhancing neutral lipid accumulation and inhibiting LD budding (Table 1), and each mutation of the eight genes yielded a modest increase (1.2-to 1.6-fold) in the LD size compared to no mutation of the WT strain ( Figure 3B and Supplementary Table 1 In terms of increasing the number of LDs, we overexpressed loa1 or deleted erd1 in the WT strain, and WT-LOA1 and WT-ΔERD1 strains were generated, respectively. We note that Erd1 has been predicted as a membrane protein for luminal ER protein retention 4 , but several studies reported that deletion of its encoding gene in yeast caused not only an ER stress response, but also the increased number of LDs and content of TAGs 5,6 ; despite the pleiotropic effect, there was no other reported phenotypic alterations, which was the reason why we chose the erd1 gene for further investigation. In this study, we reconfirmed the effect of the erd1 deletion in yeast, so the LD number of the WT-ΔERD1 strain (31.5 LDs per cell) became 1.4fold larger than that of the WT strain ( Figure 3B and Supplementary Table 1).
Overexpression of loa1 (39.5 LDs per cell) was even more effective than deletion of erd1, exhibiting an approximately 1.7-fold increase in the LD number, compared to that of the WT strain.
Even though we successfully confirmed the individual gene effect on the LD size or its number in yeast, lots of genes displayed potential trade-off between the size and the number of LDs; for instance, the WT-ΔSEI1 strain was the most effective for increasing the LD size, but its LD number dramatically decreased to be less than 38% of the WT strain's. In this study, we  Table   1). In terms of increasing the volume of LDs, deletion of tgl3 was the most effective, and overexpression of lro1 and deletion of tgl4, both of which similarly affected on the increase of LD volume, was the next most effective. Based on this observation, we combined three mutations to build the LD-size strain (WT-ΔTGL3 ΔTGL4 LRO1). The design of the LDnumber strain was simple to be WT-LOA1 ΔERD1 as we evaluated the regulatory effect of only two genes (loa1 and erd1).
Indeed, compared to the WT yeast cells, our engineered LD-size and LD-number cells demonstrated much larger LDs and larger LD population, respectively ( Figure 3C and